Questions about RoKSN and RoKSN genotyping

[Mingwei]

To improve efficiency in breeding with rugosas and species roses, I plan to add RoKSN genotyping in my breeding work. Before starting the lab works, I have several questions:

1. I plan to order primers from these articles

Kawamura, K., Ueda, Y., Matsumoto, S., Horibe, T., Otagaki, S., Wang, L., Wang, G., Hibrand-Saint Oyant, L., Foucher, F., Linde, M., & Debener, T. (2022). The identification of the Rosa S-locus provides new insights into the breeding and wild origins of continuous-flowering roses. Horticulture research, 9, uhac155. https://doi.org/10.1093/hr/uhac155

Soufflet-Freslon, V., Araou, E., Jeauffre, J., Thouroude, T., Chastellier, A., Michel, G., Mikanagi, Y., Kawamura, K., Banfield, M., Oghina-Pavie, C., Clotault, J., Pernet, A., & Foucher, F. (2021). Diversity and selection of the continuous-flowering gene, RoKSN, in rose. Horticulture research, 8(1), 76. https://doi.org/10.1038/s41438-021-00512-3

Bai, M., Liu, J., Fan, C., Chen, Y., Chen, H., Lu, J., Sun, J., Ning, G., & Wang, C. (2021). KSN heterozygosity is associated with continuous flowering of Rosa rugosa Purple branch. Horticulture research, 8(1), 26. https://doi.org/10.1038/s41438-021-00464-8

Anyone have any experiences on these primers? Is it able to repeat the result using the information from the source above? And any other primer sets or sources proved reliable?

2. Many roses with good reblooming, like some Hybrid Musks and Noisettes, are proved not homozygous of ksn^copia or ksn^null. So I think that roses with homozygous MUST be reblooming ones, but ones with heterozygous of ksn and KSN can be once blooming to any degree of reblooming based on other genes, genetic background or other factors. Does it sounds reasonable?

3. Almost all typical rugosa roses are not homozygous of ksn, many of them are reblooming (perhaps based on KSN^A181 genotype). I have a hybrid rugose seedling with juvenile blooming (or almost) and good reblooming like an HT, but about a dozen of its siblings provide only vegetative growth this year. All of them are impossible to be homozygous in ksn in theory according to their parents. So it is meaningful to create a rugosa with homozygous ksn to improve reblooming? Anyone have any experience on that?

4. Is there any hybrid rugosa varieties can provide seedlings with juvenile blooming according to your experiences?

I know discussing such topics might be boring, but I really need such help. If I can get any result, I’m glad to share if helpful.

[SeasideRooftop]

Hi Mingwei,
I can’t help with your first questions, but last year I asked basically the same question as your question 4, and you might find some info in that thread:

Best rugosas for juvenile bloom?

Hi everyone, Call me impatient but I would like to use rugosas that offer a decent percentage of juvenile blooming seedlings. I added Will Alderman early this year, since I had read on this forum that he does have the genes for it. He’s still new so there were relatively few flowers to work with this spring, but a few took. I can also say from my experiments last year that Jean de Luxembourg gave me 3/4 juvenile bloomers, but that one barely counts as a rugosa (at least in appearance, parenta…

[jeekinlau]

Hi Mingwei,

You can correct me if I am wrong, but in the 9 years I spent at the Texas A&M University rose breeding program, I don’t really see a need for using markers for everblooming. At least from a breeding perspective. If your seedlings dont bloom early on within the first 2-3 months, at least when it is 6 to 12 inches big, then it’s probably a once bloomer. Definitely if it has not bloomed in the first 10ish months then it is a once bloomer.

From a practical breeding perspective, don’t make things more difficult for yourself by doing marker assisted breeding on traits that are easily selectable. Focus the marker assisted work on phenotypes that take longer to phenotype like disease resistance.

But if you are trying to do this work from an academic perspective, it may be reason enough to do it. But you have to ask yourself, what is the academic impact? If you develop markers, so what? Will breeders use it for an trait that is easy to select for.

Just my two cents happy to discuss further maybe I am missing something?

Best,

Jeekin Lau

[Mingwei]

Hi Jeekin

Very happy to hear your suggestions and I get what you means. To my knowledge, reblooming phenotype resulted in homozygous ksn genotype is generally linked with juvenile blooming as you mentioned, but still some weird situations puzzled me.

The rose breeding program I joined is mainly about Rugosas, which mainly served as food ingredient and material for perfume industry rather than ornamental plants. Most rugosa roses have a degree of reblooming, but for most of them there is a relative long interval between flushes and the quantity of rebloom is quite low, results in low yield and difficult to harvest. What’s more, as a rebloomer, rugosa seedlings tend to have a relatively long juvenile period before first blooming (according to my observation, at least 4 months after spouting, usually more than 6 months, most seems NEVER bloom in its first year). Since I know that rebloom in rugosas is not caused by homozygous ksn genotype as in Chinas and modern roses and ksn allele seems rare in “typical” hybrid rugosas no matter blooming habit, I’m wondering I can fix this genotype in rugosas to get more reliable rebloom and accelerate the breeding process (suppose that rugosa hips need very short time to ripe, maybe it’s possible to get 2 generations a year, or at least need less time to select seedlings). So I think whether I can use RoKSN genotyping to select proper parents with ksn allele like Thérèse Bugnet.

What’s more, I’m curiosity about MAS on disease resistant you mentioned above. I have heard that many times in an rose encyclopedia, but I think reliable horizontal disease resistance is caused by combination of many genes, most of them could be unknown. So if I do this, is it likely to select something with unstable vertical, race-specific resistance like ‘Baby love’?

Yours sincerely,

Mingwei Zhou

[jeekinlau]

Hi Mingwei,

I understand your problem now. I did not understand that you were working mainly with rugosas. Another observation that I have seen is that rugosas have quite larger hips than chinas and other garden roses (which is why i suppose it is used for perfume and food industry). The larger hips may be a limiting factor if the tree is pushing more energy towards the fruit, i guess it may have less energy to push towards new flowers.

MAS has been used widely in the big agronomic crops like corn and soy but roses have relatively little genomic resources. Rather than a MAS selection approach, have you considered using Genomic Selection? Because things like disease resistance and other quantitative traits are controlled by many large and small effect loci, GS would allow you to take into account all the large and small effect loci. Now you would need a reliable and cost efficient genotyping platform that has less errors than GBS. Currently we only have the affymetrics 68k snp array which is costly ~$90 a sample and 96 samples minimum.

MAS is good and for more qualitative traits or single gene traits. Although I have seen cases where MAS developed for one breeding program is really only good for that program and once it is moved out to someone else’s program, the marker does not behave the same due to the other genetic background factors playing a role.

I have moved away from rose and now work on peach crop and would love an everblooming peach where the KSN is knocked out and that would expand my crossing season by months.

Best,

Jeekin

[Mingwei]

Thanks a lot for your advices!

@jeekinlau

Rather than a MAS selection approach, have you considered using Genomic Selection?

Now I haven’t take that in consideration because the aim of my project is breeding rather than basic scientific research. Although now Next Generation Sequencing is relatively cheap, my lab do not have the platform to handle NGS datasets, do analysis like SNPs calling and GWAS, and I’m not proficient in bioinformatics (if let companies to do that, the cost must be terrible). Since several rose genomes with good quality are published, I think the genetic basis of most traits are unclear now. I’m just looking forward to more results which might useful to the breeding process.

@jeekinlau

The larger hips may be a limiting factor if the tree is pushing more energy towards the fruit, i guess it may have less energy to push towards new flowers.

I think the reblooming trait of R. rugosa is very interesting. China roses (and modern roses derived from them) are repeat blooming shrubs, their ancestor, R. chinensis var. spontanea, is a strictly once blooming, giant climber, reblooming and related traits like juvenile blooming and shrubby habit are due to mutation preserved by cultivation. However, I think the reblooming trait of R. rugosa seems at least have a low allele frequency in the natural population (I checked Flora of China and other relative sources, they do not mention the repeat habit), and some individuals like R. laxa can rebloom too. Since the rebloom rugosa seedlings do not show evidently juvenile blooming and the hips ripe very early (ensure hips from later blooms can ripe before winter), the reblooming trait of these species seems at least not a severe disadvantage in the natural.

@jeekinlau

I have moved away from rose and now work on peach crop and would love an everblooming peach where the KSN is knocked out and that would expand my crossing season by months.

I’m not familiar with fruits, but I think that might be more difficult compared to roses, raspberries and strawberries. Although stimulated by winter chill, flower initiation and blooming of once blooming roses is continuous in one growing season. But for drupes they form flower buds in late summer or autumn, and bloom in early spring. So anyway the mechanism might be more complex than that in roses. Some varieties of ornamental cherries can bloom a lot in autumn, but I would not term that as everblooming.

For spring flowering wooden plants, there is a research article reported an everblooming mutant of Chinese tulip tree (Liriodendron chinense, family Magnoliaceae). Its growing pattern is very close to reblooming roses. You can take a look if interested.

https://doi.org/10.1038/s41438-021-00610-2.

Yours sincerely

Mingwei Zhou